ISSN 0015-5497, e-ISSN 1734-9168 Folia Biologica (Kraków), vol. 64 (2016), No 4 Ó Institute of Systematics and Evolution of Animals, PAS, Kraków, 2016 doi:10.3409/fb64_4.225 Genetic Variation in the ND1 Gene and D-loop in Protected and Commercially Exploited European Cisco (Coregonus albula L.) Populations* Lucyna KIRCZUK, Anna RYMASZEWSKA, Ma³gorzata PILECKA-RAPACZ, and Józef DOMAGA£A Accepted September 15, 2016 Published December 2016 KIRCZUK L., RYMASZEWSKA A., PILECKA-RAPACZ M., DOMAGA£A J. 2016. Genetic variation in the ND1 gene and D-loop in protected and commercially exploited European cisco (Coregonus albula L.) populations. Folia Biologica (Kraków) 64: 225-233. The European cisco (Coregonus albula L.) is a species with high environmental requirements. The deterioration of environmental conditions in recent decades has decreased its distribution. Currently the species is conserved by stocking, and the few existing natural populations are at risk of extinction. Therefore, contemporary studies involve not only reporting phenotypic parameters, but also determining the genetic structure of the population. This is an important aspect monitored in the C. albula population, which provides information valuable for proper fishing economy. This study included valuable populations from lakes located in Drawa National Park (DNP) and Wigry National Park (WNP), as well as lakes used for commercial fishing. In order to molecularly characterize the European cisco, the control region and ND1 gene were sequenced from 48 individuals from 9 populations from lakes throughout northern Poland. Analysis revealed that populations from two park lakes (Marta, Ostrowieckie) are unique. This was also the case for some sequences originating from Lake Wigry. The mean value of genetic diversity was 0.2% within each region and 0.1-0.3% between the investigated regions. The obtained results demonstrated the necessity to strengthen and protect natural populations of the European cisco, which constitute a valuable element of the European ichthyofauna. Key words: cisco, Coregonus albula, mtDNA fish, sequencing. Lucyna KIRCZUK, Ma³gorzata PILECKA-RAPACZ, Józef DOMAGA£A, Department of General Zoology, University of Szczecin, Felczaka 3c, 71-415 Szczecin, Poland. E-mail: [email protected] Anna RYMASZEWSKA, Department of Genetics, University of Szczecin, Felczaka 3c Street, 71-415 Szczecin, Poland. The European cisco (Coregonus albula L.) is populations focused on growth rate, age structure a valuable species of lake ichthyofauna in Poland (CZERNIEJEWSKI et al. 2006; KOZ£OWSKI et al. (CZERNIEJEWSKI &WAWRZYNIAK 2006), Fin- 2010) and fertility (CZERNIEJEWSKI &WAWRZY- land (VILJANEN et al. 2004), England (WINFIELD NIAK 2006). Moreover, stocking activities have et al. 2004) and Lithuania (KAUPINIS &BUKEL- been conducted for many years in order to con- SKIS 2010). Due to high environmental require- serve the existing populations and stock the endan- ments for occurrence and reproduction, as well as gered populations to ensure that appropriate recently deteriorating environmental conditions, resources are available for commercial use (MIC- the geographical range of the population is being KIEWICZ &WO£OS 2012; WO£OS et al.2016). To reduced (BERNOTAS 2002). Also in Poland, in date, few studies have investigated the populations which it is a valuable component of the lake of C. albula from a genetic perspective. This is im- ichthyofauna, the species is threatened with ex- portant for assessment of the genetic diversity of tinction (WITKOWSKI et al. 2009), and the number the fish, so that excessive catches do not lead to in- of European cisco individuals caught over the past breeding and a decrease in population fitness. four decades has decreased by half (MICKIEWICZ Limiting the size of natural C. albula populations 2012). Previous studies of indigenous Polish in single lakes can lead to the elimination of ge- _______________________________________ *Co-financed by the EU Operational Programme “Sustainable Development of the Fisheries Sector and Coastal Fishing Areas 2007-2013”, contract no. 00001-61724-OR1600004/10. 226 L. KIRCZUK et al. netic variation due to genetic drift, which in turn control region, was conducted along with an negatively affects the fitness of the species. There- analysis of relationships based on these two genes. fore, monitoring allows the assessment of the ge- The study is in line with current European trends netic resources of fish in specific water bodies in research on endangered populations (BRZUZAN (PAMMINGER-LAHNSTEINER et al. 2009, WINKLER 2000; JACOBSEN et al. 2012; KUCINSKI et al. et al. 2011), and consequently provides data for ra- 2015). The analysis provided information on the tional stocking policy. According to PAMMIN- genetic variability of the European cisco popula- GER-LAHNSTEINER et al. (2009) current studies tions and on interpopulation relatedness, and may should follow this direction. be a basis for reintroduction policy. To date, Euro- Our analysis covered very valuable natural pean cisco populations from most of the investi- populations from the strict protection zone of two gated lakes have not been studied. It is of particular national parks, as well as populations from lakes significance for the restoration of the genetic included in the Natura 2000 network. One of them, structure of the European cisco in lakes whose Lake Wigry, is listed by the International Union populations have decreased or have become ex- for Conservation of Nature (IUCN) among the tinct. The aim of the study was to analyse the world’s most valuable water bodies (Project Aqua, mtDNA sequences of protected populations of Wigry National Park 2000-2016a). This is a par- C. albula from lakes located in Drawa National ticularly interesting area of investigation, as it will Park and heavily populated, commercially utilized elucidate the genetic diversity of the populations populations of north-western Poland (Western from lakes located in protected areas. Pomerania) with reference to a population widely recognized as the source population, originating The European cisco populations are found in from Wigry National Park (north-eastern region of lakes which are a natural habitat of this species. Poland). The lakes (so-called cisco lakes), have a sand and gravel bottom, and are of 1st and 2nd class purity (CZABAN 2008). The occurrence of the European Material and Methods cisco is closely related to the different intensity of fishing pressure. Before 1990 (date of establish- ment of Drawa National Park – DNP), the popula- Study sites and amplification of biological material tions of the lakes of DNP (P³ociowe, Ostrowieckie, Marta) were fished for commercial purposes. Cur- Between 7 and 10 individuals of the European rently, the lakes are situated in the strict protection cisco (C. albula) representing populations of each zone and fishing is prohibited. The populations are of the 9 investigated lakes of northern Poland were small (personal communication from DNP em- analyzed. The investigated area included the fol- ployees), and in order to protect and renew them, lowing lakes: (i) areas near the Poland-Germany stocking was conducted in 2004-2008 using mate- border: Lake Miedwie (M) and Lake Morzycko rial from spawning fish from populations of the (R) – Region I; (ii) the strict protection zone of lakes of DNP. For over 80 years, the population of Drawa National Park (DNP): Lake Ostrowieckie Lake Wigry (Wigry National Park – WNP) has (T), Lake Marta (E), Lake P³ociowe (C)–Re- been maintained by stocking using material from gion II; (iii) the centre of Western Pomerania: spawning fish from that lake. In the 1990s, the lake Lake Drawsko (D), Lake ¯erdno (Z) and Lake By- would periodically lose part of its European cisco tyñ (B) – Region III, and (iv) Lake Wigry located population as a result of eutrophication. Fish were in Wigry National Park in north-eastern Poland – also stocked in Lake Miedwie and Lake Morzycko Region IV (Fig. 1). European cisco individuals using material originating from each of the lakes. from the reservoirs of DNP were caught with per- The European ciscoes from Lake Bytyñ, Lake mission of the Ministry of the Environment, no. Drawsko and Lake ¯erdno are caught commer- DLPpn-4102-229/17717/13/M. As for other lakes, cially and their reintroduction involves material the fish were bycatch in commercial fishing. The from spawning fish from the same lake or other study involved taking muscle samples using a ster- ile technique, which were subsequently stored in lakes (personal communication from PZW SZCZECIN Eppendorf-type tubes and frozen until analysis. 2014; MODEHPOLMO 2013). To date, very few studies of the European cisco DNA extraction and mtDNA amplification have been conducted in Poland and have regarded only a few populations caught in commercially ex- (ND1 gene and D-loop) ploited lakes. The studies were based on mtDNA DNA was extracted using phenol-chloroform analysis (ND1, D-loop, ND3/4) (BRZUZAN &CIE- following BERNATCHEZ et al. (1988), and kept SIELSKI 2002; BRZUZAN et al. 2004). In this study, at -70°C until analysis. Molecular analyses in- an analysis of two mtDNA regions, i.e. ND1 volved two mtDNA fragments, i.e. the ND1 gene (NADH dehydrogenase, subunit 1) and the D-loop (NADH dehydrogenase, subunit 1 (complex I)) Sequence of ND1 and D-loop in European Cisco 227 Fig.1. Map of sampling locations in Poland. Research area: Lake Miedwie (M) and Lake Morzycko (R) – Region I; strict protection zone of Drawa National Park (DNP): Lake Ostrowieckie (T), Lake Marta (E), Lake P³ociowe (C) – Region II; centre of Western Pomerania: Lake Drawsko (D), Lake ¯erdno (Z) and Lake Bytyñ (B) – Region III, and Lake Wigry located in Wigry National Park (WNP) in north-eastern Poland – Region IV. amplified using the primer pair Tt-ND1-F1 and identical sequences within a given population Tt-ND1-R1 (approx. 1100 bp; PAMMINGER- were collapsed. LAHNSTEINER et al. 2009), and a non-coding con- The sequencing was outsourced to Macrogen trol region (D-loop) amplified using the primer Inc.